Although the precise nature of the danger signal that activates innate immunity in the acetaminophen-exposed liver is uncertain, there is no question that danger signaling is involved in the process. This is evident from studies in a mouse strain with a mutation in Tlr4
, in which liver disease is significantly attenuated following an acetaminophen challenge (14
). Recently, acetaminophen toxicity was shown to be reduced in mice that lack the ability to respond to IL-1 (15
). This observation is of interest because IL-1 secretion typically requires the combined activity of TLRs and NLRs, the former to stimulate expression of the IL-1 propeptide and the latter to process the propeptide into a mature cytokine. It also suggests a need for two, possibly unique, danger signals in the liver: one to activate a TLR and another to signal NLR assembly into an inflammasome (6
If the danger signal that augments acetaminophen-induced liver injury is not HMGB1, then another molecule that could accomplish this task is DNA from dying hepatocytes. DNA interacts specifically with TLR9, which, like all nucleic acid–sensing TLRs, is sequestered intracellularly within endosomes. TLR9 was once considered incapable of binding mammalian DNA because of its affinity for unmethylated CpG motifs characteristic of microbial DNA. DNA from injured mammalian cells, however, has the capacity to activate TLR9 (8
), and recently even normal mammalian DNA has been shown to engage this receptor and stimulate an immune response (17
). In this issue of the JCI
, Imaeda and colleagues (18
) demonstrate that DNA is indeed a trigger of the innate immune response that amplifies acetaminophen toxicity. They showed, in a mouse model, that acetaminophen-induced liver injury is dependent upon not only IL-1β, but also IL-18, the two cytokines classically processed by caspase-1 (Figure ). They determined that acetaminophen-mediated induction of pro–IL-1β and pro–IL-18 mRNA in the liver is Tlr9 dependent; in addition, they showed that cleavage of the IL-1β propeptide to the mature cytokine in the acetaminophen-treated liver requires the presence of the Nalp3 inflammasome. Inhibition of either Tlr9 signaling or Nalp3 activity by genetic or pharmacologic means markedly attenuated liver injury and improved survival in acetaminophen-treated mice. These results indicate that Tlr9 and Nalp3 must both be functional to activate the innate immune system following acetaminophen exposure, and they underscore the degree to which immune-mediated collateral damage intensifies drug-induced liver injury.
DNA-mediated danger signaling in acetaminophen toxicity.
Imaeda et al. (18
) did not demonstrate that DNA released specifically from dying hepatocytes is the stimulus for Tlr9 activation in the liver following acetaminophen treatment. They did, however, demonstrate that DNA from dead (UV-irradiated) hepatocytes upregulates IL-1β in a Tlr9-dependent manner when infused into a normal liver in vivo. They also made the unique observation that much of the innate immune response to acetaminophen poisoning is mediated by sinusoidal endothelial cells (SECs), which are actively endocytic cells. TLR9 is expressed by several resident liver cells, including hepatocytes, SECs, Kupffer cells, and stellate cells, but among these, SECs exhibit the greatest potential for taking up extracellular DNA (19
). Purified SECs displayed DNA-mediated activation of Tlr9 and acetaminophen-mediated activation of caspase-1 (18
), which pinpoints these cells as immune effectors in acetaminophen toxicity and places them in a category with bone marrow–derived cells as sites of the molecular machinery of the inflammasome.
Viewing immune amplification of acetaminophen-induced liver injury as a therapeutic target, Imaeda and colleagues subsequently investigated whether the immune-mediated collateral damage in an acetaminophen-treated liver was preventable by administration of an antiinflammatory agent (18
). Aspirin, when given before acetaminophen, significantly protected mice against acetaminophen-induced liver injury. Indeed, aspirin-pretreated mice fared as well or better after acetaminophen challenge than mice lacking Tlr9 or components of the Nalp3 inflammasome. The protective effect of aspirin coincided with reduced induction of hepatic pro–IL-1β and pro–IL-18 mRNA after acetaminophen administration. This suggests that aspirin is exerting its protective effect on the Tlr9 arm of the danger pathway during acetaminophen toxicity, perhaps by inhibiting IκB kinase β (20
). Aspirin also inhibited experimental inflammation induced by monosodium urate crystals, which activate Nalp3, but it remains unclear whether this is interpretable as an independent effect of aspirin on caspase-1 activation by the Nalp3 inflammasome (Figure ).